1. Field of The Invention
The present invention relates to a method for purifying a gene-expression product produced by recombinant DNA technique. More particularly, the present invention is concerned with a method for purifying useful substances which are obtained from a culture of transformants prepared by recombinant DNA technique, by which allergen derived from the transformants and inevitably contained in the useful substances is effectively removed. By the method of the present invention, useful substances produced biologically can be efficiently purified to such an extent that no allergen is detected by any customary analysis. The method of the present invention is especially useful for the purification of active ingredients for drugs and quasi-drugs to be directly administered or applied to a human body. For example, the present method is extremely useful in the preparation of vaccine antigens, enzymes for pharmaceutical use, enzymes for toothpaste and toothpowder, proteins for cosmetics, etc.
2. Discussion Of Related Art
When a living body is antigenically stimulated by inoculation or inhalation of an antigen, the living body produces an antibody which specifically reacts with the antigen. Thereafter, if the same kind of antigen as the above-mentioned antigen enters the living body, an antigen-antibody reaction occurs in the living body, causing the antigen to be destroyed as a foreign matter. This is a protection mechanism against infections and called an immunity phenomenon. In some cases, however, upon entrance of an antigen, there occurs an abnormal or hypersensitive reaction, such as local inflammation or a sudden shock which is quite different from the immunity phenomenon. This abnormal reaction is called an allergic reaction. At present, the allergic reaction is classified into four types, i.e. Types I, II, III and IV, according to the manifestation of the allergic reaction and the condition of the patient. Among the four types, Type I is well known as a major allergic reaction and is called immediate-type allergy or anaphilaxis. In the cases of human bodies, it is said that Type I allergy is generally caused by immunogloblin E (IgE) antibody. Illustratively stated, if a human body is stimulated by an allergen as an antigen, IgE antibody against the allergen is produced in the body and carried thereby. Thereafter, if the same kind of allergen as mentioned above enters the body, an antigen-antibody reaction occurs between the allergen and the IgE antibody, thereby causing an allergic reaction. Substances which are known as allergen or sources of allergen are, for example, pollens, insects, skin fragments, mold, substances derived from microorganisms, foods, drugs, chemical, etc. In general, the amount of IgE antibody carried by human bodies, which is reactive to such substances, can be measured by a passive cutaneous anaphylaxis (PCA) reaction test.
Contamination of vaccine injections and the like, which are administered directly to human bodies, with allergen as an impurity greatly impairs medicines which must be safe. However, in the preparation of biological medicines such as a vaccine, there are still many requirements and problems to be studied concerning activity, safety and homogenity, and, therefore, the investigations currently conducted in the field has not yet been extended to development of a high-order purification technique giving priority especially to the removal of allergen. [See, for example, Methods in Microbiology, vol. 5B, pp. 425-454 (1971), Academic Press, England; Proc. Natl. Acad. Sci., 76, 5601-5605 (1979); and Nucleic Acids Research, 11, 2745-2763 (1983).]
Recently, there has been made an attempt to reduce the IgE antibody inducing ability of allergen by modifying allergen with polyethylene glycol, pullulan, dextran, a polyamine, a fatty acid, urea, glutaraldehyde, formalin, etc. However, this study is still at the basic stage.
Incidentally, the rapid development of the genetic engineering in recent years has made it possible to prepare transformants of microorganisms and somatic cells and to produce various useful substances from cultures of such transformants. On the other hand, such useful substances are likely to be contaminated with allergen derived from the transformants. However, in the field of genetic engineering also, importance is not yet placed on the high-order purification for removing allergen from gene-expression products contaminated therewith. Although affinity chromatography in which a monoclonal antibody is used, large-scale electrophoresis, etc. have been developed for purifying gene-expression products, they are not so effective for removing allergen derived from the transformants. Further, the purification by affinity chromatography has drawbacks that since extremely low pH values are employed for eluting the adsorbed product, the proteinous product to be purified is liable to be denatured, resulting in low immunogenicity of the product and that when the desired product adsorbed in the column is eluted, the product is necessarily contaminated by the antibody, so that an additional purification for removing the antibody is needed. On the other hand, in the purification by electrophoresis, there are also various disadvantages, such as restriction of employable medium, susceptibility of purification degree to the conditions of the operation, so that electrophoresis is not suited for a large-scale purification.
Besides the above-mentioned purification methods, the purification by gel filtration and ion exchange column chromatography have been proposed, but they are also defective. For example, in the case where a crude product containing the surface antigen of hepatitis B virus (HBs antigen) is purified by gel filtration, the yield of HBs antigen is very low, for example as low as 5 to 20%, and the HBs antigen is liable to be inactivated in the column due to the low concentration of HBs antigen. In the case where a crude product containing HBs antigen is purified by ion exchange column chromatography, the purity of the product after the purification is insufficiently low.
In connection with the above-mentioned customary purification methods, reference can be made to the following:
International Patent Application (PCT) Publication No. WO85/05631 PA0 European Patent Application Publication No. 0 140 386 PA0 U.S. Pat. No. 4,503,035 PA0 U.S. Pat. No. 4,505,893 PA0 U.S. Pat. No. 4,508,709 PA0 U.S. Pat. No. 4,508,833 PA0 U.S. Pat. No. 4,514,506 PA0 U.S. Pat. No. 4,525,459 PA0 U.S. Pat. No. 4,552,758 PA0 U.S. Pat. No. 4,554,157 PA0 U.S. Pat. No. 4,565,697 PA0 U.S. Pat. No. 4,578,269
Accordingly, in the field of the genetic engineering including the biological production of medicines, it is of urgent necessity to establish a high-order purification technique for removing allergen derived from transformants and contained in gene-expression products.